TY - JOUR
T1 - Evidence Supporting Catalytic Roles for Aspartate Residues in Phosphoribulokinase
AU - Charlier, Henry A.
AU - Runquist, Jennifer A.
AU - Miziorko, Henry M.
PY - 1994/8/1
Y1 - 1994/8/1
N2 - The DNA encoding Rhodobacter sphaeroides phosphoribulokinase (PRK) has been modified to allow ligation into pET-3d. Using the resulting expression plasmid, PRK was overexpressed in Escherichia coli and isolated in milligram quantities. Homogeneous preparations of the enzyme exhibit properties comparable to those of PRK expressed using a previously described pUC19-derived construct [Sandbaken et al., Biochemistry 31, 3715–3719]. Mutagenesis experiments have been designed to produce conservative substitutions that eliminate the carboxyl groups of each of four conserved acidic residues (D42, E131, D169, and E178). Using the newly developed expression system, the resulting PRK variants have been expressed, isolated, and characterized. Expression levels and recoveries upon affinity chromatography purification are similar to the results obtained with wild-type PRK. Apparent substrate affinities of these mutant proteins do not differ greatly from values observed for wild-type PRK. In contrast, these PRK variants display a wide range of Vmaxvalues, ranging from wild-type activity (∼200 units/mg; E178A) to levels that are diminished by 4 (D169A) to 5 (D42A, D42N) orders of magnitude. That the large diminutions in catalytic activity are significant and do not merely reflect gross perturbations in protein structure is suggested not only by the modest effects on substrate affinity but also by the allosteric properties of D169A, D42A, and D42N. The activities of these proteins, like that of wild-type PRK, are markedly stimulated by the positive effector NADH. The magnitude of the Vmaxperturbations suggests that D42 and D169 are candidates for the role of active site base or activator cation ligand. In contrast to the marked diminution of Vmaxobserved upon mutation of D42 or D169, only a 2 order of magnitude decrease is observed with E131A; much of this effect may be attributed to the fact that this variant no longer is sensitive to allosteric stimulation by NADH.
AB - The DNA encoding Rhodobacter sphaeroides phosphoribulokinase (PRK) has been modified to allow ligation into pET-3d. Using the resulting expression plasmid, PRK was overexpressed in Escherichia coli and isolated in milligram quantities. Homogeneous preparations of the enzyme exhibit properties comparable to those of PRK expressed using a previously described pUC19-derived construct [Sandbaken et al., Biochemistry 31, 3715–3719]. Mutagenesis experiments have been designed to produce conservative substitutions that eliminate the carboxyl groups of each of four conserved acidic residues (D42, E131, D169, and E178). Using the newly developed expression system, the resulting PRK variants have been expressed, isolated, and characterized. Expression levels and recoveries upon affinity chromatography purification are similar to the results obtained with wild-type PRK. Apparent substrate affinities of these mutant proteins do not differ greatly from values observed for wild-type PRK. In contrast, these PRK variants display a wide range of Vmaxvalues, ranging from wild-type activity (∼200 units/mg; E178A) to levels that are diminished by 4 (D169A) to 5 (D42A, D42N) orders of magnitude. That the large diminutions in catalytic activity are significant and do not merely reflect gross perturbations in protein structure is suggested not only by the modest effects on substrate affinity but also by the allosteric properties of D169A, D42A, and D42N. The activities of these proteins, like that of wild-type PRK, are markedly stimulated by the positive effector NADH. The magnitude of the Vmaxperturbations suggests that D42 and D169 are candidates for the role of active site base or activator cation ligand. In contrast to the marked diminution of Vmaxobserved upon mutation of D42 or D169, only a 2 order of magnitude decrease is observed with E131A; much of this effect may be attributed to the fact that this variant no longer is sensitive to allosteric stimulation by NADH.
UR - http://www.scopus.com/inward/record.url?scp=0028128635&partnerID=8YFLogxK
U2 - 10.1021/bi00197a039
DO - 10.1021/bi00197a039
M3 - Article
C2 - 7914091
AN - SCOPUS:0028128635
SN - 0006-2960
VL - 33
SP - 9343
EP - 9350
JO - Biochemistry
JF - Biochemistry
IS - 31
ER -